ca - and protein kinase c-dependent signaling pathway for

Ca2�- and Protein Kinase C-dependent Signaling Pathway forNuclear Factor-�B Activation, Inducible Nitric-oxide SynthaseExpression, and Tumor Necrosis Factor-� Production inLipopolysaccharide-stimulated Rat Peritoneal Macrophages*

Received for publication, March 23, 2006, and in revised form, August 21, 2006 Published, JBC Papers in Press, August 21, 2006, DOI 10.1074/jbc.M602739200

Xueyuan Zhou, Wenxiu Yang1, and Junying LiFrom the Department of Biophysics in the School of Physics, Key Laboratory of Bioactive Materials of Education Ministry,Nankai University, Tianjin 300071, Peoples Republic of China

Lipopolysaccharide (LPS)-activated macrophages are pivotalin innate immunity. With LPS treatment, extracellular signalsare transduced into macrophages via Toll-like receptor 4 andinduce inflammatory mediator production by activating signal-ing pathways, including the nuclear factor-�B (NF-�B) pathwayand the mitogen-activated protein kinase (MAPK) pathway.However, the mechanisms by which the intracellular free Ca2�

concentration ([Ca2�]i) increases and protein kinase C (PKC) isactivated remain unclear. Therefore, we investigated the signal-ing pathway for Ca2�- and PKC-dependent NF-�B activation,inducible nitric-oxide synthase expression, and tumor necrosisfactor-� (TNF-�) production in LPS-stimulated rat peritonealmacrophages. The results demonstrated that the LPS-inducedtransient [Ca2�]i increase is due to Ca2� release and influx.Extracellular and intracellular Ca2� chelators inhibited phos-phorylation of PKC� and PKC�. A PKC�-specific and a generalPKC inhibitor blunted phosphorylation of serine inmitogen-acti-vated/extracellular signal-regulated kinase kinase kinase (MEKK)1.Moreover, aMEKKinhibitor reducedactivationof inhibitory�Bkinase and NF-�B. Upstream of the [Ca2�]i increase, a protein-tyrosine kinase inhibitor reduced phosphorylation of phospho-lipase C (PLC) �. Furthermore, a PLC inhibitor eliminated thetransient [Ca2�]i increase and decreased the amount of activatedPKC. Therefore, these results revealed the following roles of Ca2�

and PKC in the signaling pathway for NF-�B activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosinekinasemediates PLC�1/2 phosphorylation, which is followed by a[Ca2�]i increase. Several PKCs are activated, and PKC� regulatesphosphorylation of serine inMEKK1. Moreover, MEKKs regulateinhibitory �B kinase activation. Sequentially, NF-�B is activated,and inducible nitric-oxide synthase and tumor necrosis factor-�production is promoted.

Lipopolysaccharide (LPS),2 one of the membrane compo-nents of Gram-negative bacteria, induces a variety of responses

to severe infection, such as local inflammation, antibody pro-duction, and septic shock.Macrophages respond to LPS early inthe infection and thus play a pivotal role in host defense. How-ever, at high concentrations, LPS stimulates macrophages torelease massive amounts of pro-inflammatory mediators suchas interleukins (ILs), tumor necrosis factor � (TNF-�), super-oxide, and nitric oxide (1, 2). These pro-inflammatory media-tors can disturb normal cellular function, and this disruptioncan lead tomultiple organ dysfunction syndromes or lethal sep-tic shock (3, 4). Therefore, the investigation of LPS-inducedsignaling pathways in macrophages is important and necessaryfor discovering potential therapeutic targets and drugs.In recent years, it has been found that LPS binds with LPS-

binding protein and interacts with a complex that consists ofCD14, MD2, and Toll-like receptor 4 (TLR4); the complextransduces the signal generated by its interaction with LPS intothe interior of the cell (5, 6). The intracellular Toll/IL-1 recep-tor domain of TLR4 recruits myeloid differentiation factor 88,IL-1 receptor-associated kinase, myeloid differentiation factor88 adaptor-like protein, Toll/IL-1 receptor adaptor protein,and TNF receptor-associated factor 6 (TRAF6) to form a com-plex, which can transduce the signal further. Several signalingpathways are activated sequentially, including the mitogen-ac-tivated protein kinase (MAPK) pathway and the nuclear fac-tor-�B (NF-�B) pathway (7).

In LPS-stimulated macrophages, MAPKs are activated by athree-tiered cascade of kinases: 1) mitogen-activated/extracel-lular signal-regulated kinase kinase kinase (MEKK); 2) MAPKkinase (MEK); 3) MAPKs, including p38, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase(JNK). MAPKs can activate some transcription factors, such asactivator protein 1 and activating transcription factor 2, andplay important roles in the production of inflammatory medi-ators (8–11). However, how does LPS activate theMAPK path-

* The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed. Tel.: 86-22-23501005; Fax:86-22-23501594; E-mail: [email protected].

2 The abbreviations used are: LPS, lipopolysaccharide; IL, interleukin; TNF-�,tumor necrosis factor �; TLR4, Toll-like receptor 4; TRAF, TNF receptor-associ-ated factor; MAPK, mitogen-activated protein kinase; NF-�B, nuclear factor-�B;MEKK, mitogen-activated/extracellular signal-regulated kinase kinase; MEK,

mitogen-activated/extracellular signal-regulated kinase kinase kinase; IKK, I�Bkinase; PKC, protein kinase C; PTK, protein-tyrosine kinase; I�B, inhibitory �B;TAK, transforming growth factor �-activated kinase; TAB, TAK1-binding pro-tein; [Ca2�]i, intracellular free Ca2� concentration; Ins(1,4,5)P3, inositol 1,4,5-triphosphate; iNOS, inducible nitric-oxide synthase; PLC, phosphatidylinosi-tol-specific phospholipase C; Fura-2/AM, Fura-2 acetoxymethyl ester; PMSF,phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; HRP, horse-radish peroxidase; PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; DAG,1,2-diacylglycerol; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N�,N�-tet-raacetic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 42, pp. 31337–31347, October 20, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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way in macrophages? Some reports have suggested that phos-phoinositide 3-kinase, protein kinase C (PKC), and someprotein-tyrosine kinases (PTKs) are responsible forMEKKacti-vation (12, 13).NF-�B regulates the expression of many cytokines in LPS-

stimulated macrophages and plays an important role in theimmune system. Recently, considerable progress has beenmade in determining the function and regulation of NF-�B. Inmacrophages, NF-�B is a p65/p50 dimer and is inactivated bybinding with inhibitory �B (I�B). The inhibitory �B kinase(IKK) �, a component of the IKK complex, can phosphorylateI�B�, and leads to the ubiquitination and degradation of I�B.Meanwhile, p65/p50 is translocated into the nucleolus (14, 15).Therefore, the activation of the IKK complex plays a crucial rolein NF-�B activation. Furthermore, two sets of adaptors arelinked to IKK activation. The first involves transforminggrowth factor �-activated kinase 1 (TAK1) and two associatedadaptors, TAK1-binding protein 1 (TAB1) and TAB2. Afterstimulation, TRAF6ubiquitylates itself or the component of theTAK1�TAB1�TAB2 complex and primes it for IKK activation.Another protein that has been reported to act as a bridgebetween TRAF6 and IKK is ECSIT. Moreover, MEKK1 andMEKK3 have been implicated in IKK activation in receptor/TLR4-mediated NF-�B activation pathways (7).Although many studies have revealed that TRAF6 is related

to MEKKs and IKK activation, the question about how TRAF6activates them is unanswered. Intracellular free Ca2� concen-tration ([Ca2�]i) is important for cellular function; however,few studies have been focused on the signaling pathway(s)involving [Ca2�]i in LPS-stimulated macrophages. Severalreports have indicated that LPS treatment can cause an increasein [Ca2�]i, which is related to TNF-� production in alveolarmacrophages and Kuffer cells (16, 17). Our previous reportshave also revealed that the transient increase in [Ca2�]i in LPS-stimulated rat peritoneal macrophages plays an important rolein TNF-� production (18). Furthermore, LPS also induces abiphasic change in inositol 1,4,5-triphosphate (Ins(1,4,5)P3) inC3H/HeN mouse macrophages, and the change leads to theactivation of a TNF-� gene. The later phase of the Ins(1,4,5)P3change ismediated by a�2 type of phosphatidylinositol-specificphospholipase C (PLC�2) (19). However, the mechanisms bywhich LPS induces a transient increase in [Ca2�]i and the roleof [Ca2�]i in NF-�B and MAPK signaling pathways have notbeen elucidated. Additionally, LPS can activate PKC isoen-zymes and regulate the expression of inflammatory mediatorsinmacrophages (20). However, which kinds of PKC isoenzymesare responsible for NF-�B activation and how they work in thesignaling pathway remain unknown.In the present study, we investigated the [Ca2�]i- and PKC-

dependent signaling pathway for NF-�B activation, induciblenitric-oxide synthase (iNOS) expression, and TNF-� produc-tion in LPS-stimulated rat peritoneal macrophages. On thebasis of the experimental results, we propose a newmode of thesignaling pathway.

EXPERIMENTAL PROCEDURES

Reagents—Dimethyl sulfoxide, LPS (Escherichia coli serotype0127:B8 prepared by phenol extraction), Fura-2 acetoxymethyl

ester (Fura-2/AM), the PTK inhibitor (genistein), the PKCinhibitor (GF109203X), the PLC inhibitor (U73122), andEGTAwere purchased from Sigma. BATPA-AM (intracellular Ca2�

chelator) was obtained from Molecular Probes. The MEKKinhibitor (PD98059), the PKC�-specific inhibitor (Go6976),and thePKC�-specific inhibitor (Go6983)were purchased fromCalbiochem. The PKC�-specific inhibitor (LY379196) wasacquired from Eli Lilly.Isolation and Culture of Macrophages—Male Wistar rats

(about 250 g), whichwere treated humanely in compliancewithinstitutional guidelines, were killed. Hanks’ balanced salt solu-tion was injected into the abdomen of each rat.Macrophages inHanks’ balanced salt solution removed from the abdomenwerecentrifuged at 200 � g for 5 min. The cell pellet was collected,and the cells were cultured in RPMI 1640 and 10% fetal bovineserum for 6 h at 37 °C in 5% CO2. Adherent cells were har-vested, re-suspended, and incubated for another 48 h beforeanalysis. Nonspecific esterase staining showed that 95% of theadherent cells were macrophages.Immunoblot Assay to Detect PLC Phosphorylation—Cells

(107) were scraped from dishes into cold lysis buffer (0.1 M Tris,pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 20 mM

NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride(PMSF), and 1�g/ml each of pepstatin and leupeptin) after theywere washed twice in cold phosphate-buffered saline (PBS).After centrifugation (10,000 � g for 10 min at 4 °C), the solublefraction, which contained PLC, was collected. The proteins inthe soluble fraction were separated by SDS-PAGE (7%) andtransferred to polyvinylidene fluoride membranes. Immuno-blotting was carried out by treating the membrane with 4.5 �gof antibody specific for activated phosphotyrosines or phos-phoserines of PLC�1 (Tyr783), PLC�2 (Tyr759), or PLC�3(Ser537) (Cell Signaling Technology; ratio of antibody dilution,1:200) for 12 h.Tonormalize the amount of sample in each lane,immunoblotting was also carried out by using 4.5 �g of poly-clonal antibody to PLC�1, PLC�2, or PLC�3 (Cell SignalingTechnology; ratio of antibody dilution, 1:200) for 12 h. Biotiny-lated antibody specific for rabbit immunoglobulin (AmershamBiosciences) was employed as the secondary antibody. Thechemiluminescence of the streptavidin-horseradish peroxidase(HRP) conjugates was detected by film.Measurement of [Ca2�]i in Macrophages—Intracellular free

Ca2� was detected by using the ratiometric fluorescent Ca2�

indicator dye Fura-2 and a microspectrofluorometer. Cellswere incubated in 3�MFura-2/AM for 50min at room temper-ature and washed twice with PBS. Changes in the fluorescenceintensity of Fura-2 at excitation wavelengths of 340 and 380 nmand the emission wavelength of 510 nm were monitored in anindividual peritoneal macrophage. [Ca2�]i was calculated byusing the following equation.

[Ca2� ]i � Kd �[Ca-Fura-2]

[Fura-2]� Kd �

F340

F380(nmol/l) (Eq. 1)

Kdwas the constant for Fura-2 chelatingCa2�, and its valuewasabout 135 nmol/liter when the temperature was 22 °C. In thepresent experiment, F340/F380 was directly related to [Ca2�]i.

[Ca2�]i and PKC Are Related to LPS-induced NF-�B Activation

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Measurement of Total PKC Activity—Total protein kinase Cactivity was determined as described by Lee and Wu (21) butwith modifications. After treatment, cells were washed twicewith PBS and scraped into cold lysis buffer (20 mM Tris-HCl,pH 8.0, 0.5 mM EDTA, 0.5 mM EGTA, 2.5 mM PMSF, 5 �g/mlleupeptin, and 5 �g/ml antipain). After collection, cells weresonicated for 10 pulses. Sonicated samples were centrifuged at15,000� g for 30min at 4 °C, and the supernatant was collectedand aliquoted (200 mg/tube). PKC activity in the supernatantwas measured immediately by using the Pierce ColorimetricPKC Assay Kit. The PKC-dependent phosphorylated peptidewas quantified by spectroscopy at 570 nm.Immunoblot Assay toDetect Phosphorylated PKC Isoenzymes—

After treatment with LPS, macrophages were washed twice inice-cold PBS. Cell pellets were resuspended in buffer (50 mMTris-HCl, pH 7.5, 150mMNaCl, 1 mM EGTA, 1mM dithiothre-itol, 1 mM PMSF, 1 mMNa3VO4, 1 mMNaF, 5 �g/ml aprotinin,5 �g/ml leupeptin, 5 �g/ml antipain, 1% Nonidet P-40, 0.25%sodium deoxycholate) for 30 min at 4 °C. Lysates were centri-fuged at 10,000 � g for 30 min at 4 °C, and the supernatantswere collected. Proteins in the supernatant were separated bySDS-PAGE (12%). SDS-separated proteins were equilibrated intransfer buffer (50 mMTris, pH 9.0–9.4, 40 mM glycine, 0.375%SDS, 20% methanol) and electrotransferred to Immobilon-Pmembranes. Nonspecific binding sites were blocked by incuba-tion for 1 h in Tris-buffered saline (10 mM Tris, 150 mM NaCl)containing 5% nonfat dry milk and 0.05% Tween 20. Themem-branes were thenwashed and incubatedwith 6�g of anti-phos-pho-PKC� (Ser657) antibody (dilution ratio, 1:5000)(SantaCruzBiotechnology), anti-phospho-PKC� (Ser660) antibody (dilu-tion ratio, 1:2500) (Santa Cruz Biotechnology), anti-phospho-PKC� (Thr505) antibody (dilution ratio, 1:1000) (Cell SignalingTechnology), or anti-phospho-PKC (Ser729) antibody (dilu-tion ratio, 1:2500) (Upstate) for 10 h at 4 °C. To normalize theamount of sample in each lane, immunoblottingwas carried outby treating the membrane with 6 �g of polyclonal antibodies toPKC� (dilution ratio, 1:5000), PKC� (dilution ratio, 1:2500),PKC� (1:500), and PKC (dilution ratio, 1:500) (Santa Cruz Bio-technology) for 10 h at 4 °C. An HRP-conjugated goat anti-mouse IgG antibody (1:20,000 dilution) was used as the second-ary antibody, and the chemiluminescence was detected on film.Immunoprecipitation and Immunoblot Analysis of Phospho-

rylated Serine in MEKK1—Cells were washed in ice-cold PBSand scraped into PBS. Cells were pelleted by centrifugation andlysed in whole cell extract lysis buffer (50 mM Tris-HCl, pH 7.9,150 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100, 10% glycerol,1.0 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 0.2 mM leupeptin,0.2 mM pepstatin A) for 30 min on ice. The lysates were centri-fuged at 10,000 � g for 10 min at 4 °C, and supernatants wereisolated. After normalization (immunoblotting assay), celllysates that contained equal amounts of total proteinwere incu-bated with 2 �g of polyclonal anti-MEKK1 antibody (43-Y,Santa Cruz Biotechnology; dilution ratio, 1:200) for 2.5 h at 4 °Cand then with 4.5 �g of protein G-conjugated agarose beads(Amersham Biosciences) for 1 h at 4 °C. After centrifugation,MEKK1 isolated from the beads was separated by SDS-PAGE(12%) and transferred onto a nitrocellulose membrane. Immu-noblot analysis was carried out by using monoclonal antibody

to phosphoserine (Santa Cruz Biotechnology; dilution ratio,1:1000). Biotinylated antibody to rabbit Ig (Amersham Bio-sciences) was employed as the secondary antibody. The chemi-luminescence of the streptavidin-HRP conjugates was detectedby film.Immunoprecipitation and IKKProteinKinase Activity Assays—

After LPS stimulation of macrophages, the cytosol wasextracted by using 200 �l of IKK CE buffer (10 mM HEPES-KOH, pH 7.9, 250 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40,0.2%Tween 20, 2mMdithiothreitol, 1mMPMSF, 20mM �-glyc-erophosphate, 10 mM NaF, 0.1 mM Na3VO4), and the proteinsin each extract were normalized using measurements fromBradford assays. Cytoplasmic extracts (100 �l) were incubatedwith 1 �g of monoclonal antibody specific for IKK� (BDPharmingen) for 2 h at 4 °C, and then with protein G-conju-gated agarose beads (Amersham Biosciences) for 1 h at 4 °C.After the beadswerewashed twicewith IKKCEbuffer and oncewith kinase buffer (20mMHEPES, pH 7.7, 100mMNaCl, 10mMMgCl2, 2 mM dithiothreitol, 1 mM PMSF, 20 mM �-glycero-phosphate, 10 mM NaF, 0.1 mM Na3VO4), they were incubatedwith 20 �l of kinase buffer containing 20 �M ATP, 10 �Ci of[32P]ATP, and 0.5 �g of bacterially expressed GST-I�B�-(1–54) substrate at 30 °C for 30 min. The reaction mixture under-went SDS-PAGE (10%), and the proteins were visualized. Tonormalize kinase activities, the proteins that appeared to be50–175 kDa were transferred to polyvinylidene fluoride mem-branes (Amersham Biosciences), and the blots underwentstandard immunoblotting techniques to detect IKK (22).Immunoblot Assay to Detect Phosphorylated I�B� in the

Cytoplasm—Cells (107) were washed twice with 5 ml of coldPBS and centrifuged at 350 � g for 5 min at 4 °C. Proteins werereleased by resuspension of the pellet in 250 �l of buffer (10 mMTris-HCl, pH 7.8, 10mMKCl, 2.5mMNaH2PO4, 1.5mMMgCl2, 1mMNa3VO4, 0.5mM dithiothreitol, 0.4mM 4-(2-aminoethyl)ben-zenesulfonyl fluoride/HCl, 2 �g/ml leupeptin, 2 �g/ml pepstatinA, and 2�g/ml aprotinin). The suspension was incubated on icefor 10 min and then homogenized at a moderate speed. Thehomogenate underwent centrifugation at 4,500� g for 5min at4 °C, and the supernatant was collected. After the proteins wereseparated by SDS-PAGE (10%) and transferred to nitrocellulosemembranes and after the nonspecific binding sites on themem-branes were blocked by incubation overnight at 4 °C with 1%bovine serum albumin, the membranes were then incubatedwith antibody specific for phospho-I�B� (Ser32) (dilution ratio,1:2000; Cell Signaling) for 12 h at 4 °C. To normalize theamount of sample in each lane, immunoblot analysis was alsocarried out by treating the membranes with 5 �g of polyclonalantibody to I�B� (dilution ratio, 1:1000; Cell Signaling) for 12 hat 4 °C. Biotinylated antibody to rabbit immunoglobulin(Amersham Biosciences) was employed as the secondary anti-body. The chemiluminescence of the streptavidin-HRP conju-gates was detected by film.Western Blot Detection of p65 in Nucleoli—Cells (107) were

washed twice with cold PBS. Nuclei were released by resus-pending the pellet in 250 �l of buffer A (10 mM Tris-HCl, pH7.8, 10 mM KCl, 2.5 mM NaH2PO4, 1.5 mM MgCl2, 1 mMNa3VO4, 0.5 mM dithiothreitol, 0.4 mM 4-(2-aminoethyl)ben-zenesulfonyl fluoride/HCl, 2 �g/ml leupeptin, 2 �g/ml pepsta-




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tin A, and 2 �g/ml aprotinin). After incubation for 10min on ice,the suspension was homogenized at a moderate speed. Nucleiwere collected by centrifugation at 4,500� g for 5min at 4 °C andresuspended in 100 �l of buffer B (buffer A adjusted to 20 mMTris-HCl, pH 7.8, 420 mM KCl, 20% (v/v) glycerol). Nuclei werethen lysed at 4 °C for 30 min with gentle agitation, debris wascleared by centrifugation at 10,000� g for 30min at 4 °C, and thesupernatant was collected. The proteins were separated by SDS-PAGE (10%) and transferred to nitrocellulose membranes. After

nonspecific binding sites were blocked by incubation overnight at4 °C with 1% bovine serum albumin, the membranes were thenincubated with polyclonal antibody specific for p65 (Santa CruzBiotechnology). To normalize the protein content of each sample,�-actin was detected with 15 �g of anti-�-actin antibody (Sigma,1:1000). Biotinylated antibody specific for rabbit immunoglobulin(Amersham Biosciences) was employed as the secondary anti-body. The chemiluminescence of the streptavidin-HRP conju-gates was detected by film (23).

FIGURE 1. LPS induced phosphorylation of PLC, a transient increase in [Ca2�]i, PKC activation, phosphorylation of serine in MEKK1, IKK activation,I�B� phosphorylation, p65 translocation, NF-�B activation, iNOS expression, and TNF-� production in rat peritoneal macrophages. A, immunoblotanalysis of phosphorylation of PLC�1, PLC�2, and PLC�3 in cells that were treated with no LPS (�) or with 10 �g/ml LPS. The antibodies were specific forphosphorylated PLC�1, PLC�2, or PLC�3. The protein content of each sample was normalized by analysis using the appropriate antibodies against PLC�1,PLC�2, or PLC�3. B, measurement of the transient [Ca2�]i increase in an individual macrophage. This experiment was repeated more than 9 times, andrepresentative results of experiments in which extracellular calcium was present (I) or absent (II) are shown. The arrows indicate the times when reagents wereadded. C, enzyme-linked immunosorbent assay (Pierce Colorimetric PKC Assay) to measure total PKC activity. D, immunoblot analysis of phosphorylated PKCisoenzymes using antibodies to phosphorylated PKC�, -�, -�, and -. The protein content of sample was normalized by immunoblot analysis with antibodiesto PKC�, -�, -�, and -. E, immunoprecipitation analysis of phosphorylated serine in MEKK1. After immunoprecipitation with antibody to MEKK1, the boundprotein was analyzed by using an antibody to phosphorylated serine. The protein content of each sample was normalized by immunoblot analysis with anantibody to MEKK1. F, results of an immunoprecipitation and kinase assay to evaluate IKK activity. Glutathione S-transferase-I�B� served as a substrate in theassay, and its phosphorylation by IKK was quantified, normalized, and plotted. G, Western blot analysis of p65 translocation into nucleoli and immunoblotanalysis of I�B� phosphorylation in the cytoplasm. The antibodies were specific for p65 and phosphorylated I�B�. The protein content of each sample wasnormalized by immunoblot or Western blot analysis with antibodies to I�B� or �-actin. H, results of a dual-luciferase activity assay to evaluate NF-�B activation.I, immunoblot analysis of iNOS expression in macrophages. The antibody was specific for iNOS. The protein content of each sample was normalized byimmunoblot analysis with an antibody to �-actin. J, results of the TNF-� assay. The control was represented by F, LPS by f.




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Transient Transfection and Dual Luciferase Assays—Trans-fection was done by using a standard procedure for calciumphosphate precipitation. Macrophages were transfected with 3�g of luciferase reporter plasmids that contained an NF-�Bgene (pNF-�B-luc) and 3 �g of pRL-TK reporter plasmids(pRL-TK-Renilla). After 48 h, samples were harvested and pre-pared for luciferase assays according to themanufacturer’s pro-tocol (Promega Corp.). Firefly and Renilla luciferase activitieswere measured in each sample using the Dual Luciferase Assay(Promega Corp.).Immunoblot Assays to Detect iNOS Expression—After LPS

treatment, macrophages (106) were lysed in SDS sample buffer(62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mer-captoethanol, 0.02% bromphenol blue) and boiled for 5 min at100 °C. Aliquots (20 �g per lane) underwent electrophoresis ina 10% polyacrylamide gel, and the proteins were transferred tonitrocellulose membranes (Schleicher & Schuell). Membraneswere treated with 10% nonfat milk for 1 h to block nonspecificbinding sites, rinsed, and incubated with rabbit polyclonal anti-body to iNOS (Santa Cruz Biotechnology) overnight at 4 °C. Tonormalize the protein content of each sample, �-actin wasdetected with 15 �g of anti-�-actin antibody (Sigma, 1:1000).The membranes were then treated with HRP-conjugated anti-

rabbit IgG (dilution ratio, 1:2000) for 1 h. Immune complexeswere detected as described (24).TNF-� Assays—Macrophages and L929 cells (mouse,

C3H/An areolar and adipose cells) were cultured in 96-wellplates. TNF-� was detected in each defined period. After col-lecting the macrophage supernatant, an equal volume ofmedium with or without reagents was added to the wells atdifferent times. Supernatants of macrophage cell cultures (100�l/well) were collected and added with actinomycin D (Sigma;final concentration, 1 �g/ml) to L929 cells. Twenty hours later,plates underwent centrifugation at 200 � g for 5 min. Thesupernatants were discarded, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (final concentration, 1mg/ml; Sigma) without phenol red was added. After 4 h, thesupernatants were discarded, and 100�l of a solution ofMe2SOand ethanol (ratio, 1:1) was added to each well. The A570 valueswere measured by an enzyme-linked immunoabsorbent detec-tor, and the quantity of TNF-� was calculated using a standardcurve for recombinant human TNF-� (Pepro Tech EC LTD)(25).Statistical Analysis—All assays were repeated a minimum of

three times, and representative results from a single assay areshown. Some results are expressed as the mean � S.E.

FIGURE 2. An increase in [Ca2�]i affected LPS-induced activation of PKC activation, serine phosphorylation of MEKK1, and activation of IKK in ratperitoneal macrophages. Macrophages received no pretreatment (�) or pretreatment that consisted of 50 �M BAPTA-AM for 1 h or 5 mM EGTA for 5 min.Subsequently, LPS (10 �g/ml) was added. A and E, enzyme-linked immunosorbent assay (Pierce Colorimetric PKC Assay) to measure total PKC activity. B and F,immunoblot analysis of phosphorylated PKC isoenzymes. The antibodies were specific for phosphorylated PKC�, -�, -�, and -. C and G, immunoprecipitationand immunoblot analysis of phosphorylated serine in MEKK1. The antibodies were specific for MEKK1 and phosphorylated serine. D and H, results of an IKKimmunoprecipitation kinase assay.




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RESULTS

LPS-induced PLC� Phosphorylation, a Transient Increase in[Ca2�]i, PKC Activation, Serine Phosphorylation in MEKK1,IKK, and NF-�B Activation, iNOS Expression, and TNF-�Production—Within 5 min of the start of LPS treatment (10�g/ml), phosphorylated tyrosine in PLC�1 and PLC�2 wasdetected in rat peritoneal macrophages, and phosphorylationreached a maximum at 10 min. Nevertheless, phosphorylationof PLC�3 was not detected (Fig. 1A). Moreover, a transientincrease in [Ca2�]iwas evoked 110 s after the start of LPS treat-ment and continued for a total of 140 s. The peak of [Ca2�]iwaslower when 5 mM EGTA was added (the extracellular Ca2�

concentration was 1.25 mM), whereas the initial rates of Ca2�

increasewere similar in the presence and absence of EGTA (Fig.1B). Therefore, the results indicate that the initial transientincrease in [Ca2�]i is due to intracellular Ca2� release, whichmay be followed by Ca2� influx from the extracellular space.Furthermore, total PKC activity increased between 0.25 and 1 hand then began to decline (Fig. 1C). The pattern of change seenin the quantities of phosphorylated isoenzymes PKC�,�, �, and was similar to the pattern seen in total PKC activity over time(Fig. 1D).Stimulation by LPS (10 �g/ml) resulted in the phosphoryla-

tion of serine inMEKK1, which was first detected at 0.25 h andincreased for the next 0.75 h. From 1 to 3 h, MEKK1 was phos-phorylated steadily (Fig. 1E). Moreover, IKK activity increasedduring the period of 1 to 3 h and then decreased (Fig. 1F). Accu-mulation of phosphorylated I�B� in the cytoplasm and trans-

location of p65 into the nucleolus also increased markedly inthe first 3 h of LPS treatment (Fig. 1G). Meanwhile, the patternof NF-�B activation in response to LPS treatment resembledthose of phosphorylated I�B� accumulation and p65 transloca-tion (Fig. 1H). Finally, iNOS expression increased dramaticallybetween 2 and 5 h of treatment (Fig. 1I). Different slightly fromiNOS expression, TNF-� production markedly increasedbetween 3 and 8 h (Fig. 1J).Role of the [Ca2�]i Increase in LPS-induced PKC Activation,

Phosphorylation of Serine in MEKK1, and IKK Activation—Toidentify the role of the [Ca2�]i increase in the signaling pathwayfor NF-�B activation, extracellular or intracellular Ca2� waseliminated, and the effect was assessed. Pretreatment of mac-rophages with 50 �M BAPTA-AM (an intracellular Ca2� che-lator) for 1 h or with 5 mM EGTA for 5 min had no effect onLPS-induced phosphorylation of PLC�1 and PLC�2 (data notshown). However, total PKC activity and phosphorylation ofPKCs, especially Ca2�-dependent phosphorylation of PKC� andPKC�, were reduced (Fig. 2A,B,E, andF).Nevertheless, the quan-tity of phosphorylated PKC� andPKC, whose phosphorylation isCa2�-independent, was not affected obviously. Moreover, LPS-induced phosphorylation of serine in MEKK1 and activation ofIKKwere inhibited (Fig. 2,C,D,G, andH). Therefore, activationofPKC� and PKC� depends on the transient increase in [Ca2�]i inLPS-stimulated rat peritoneal macrophages.Role of PKC in LPS-induced Phosphorylation of Serine in

MEKK1 and Activation of IKK—To elucidate the relationshipbetween PKC and other molecules in the signaling pathway for

FIGURE 3. The PKC inhibitor (GF109203X) and PKC� inhibitor (LY379196) reduced LPS-induced PKC activation, phosphorylation of serine in MEKK1,and IKK activation in rat peritoneal macrophages. Macrophages were not pretreated (�) or were pretreated (�) with GF109203X (1 �M) or LY379196 (30 �M)for 30 min, and then LPS (10 �g/ml) was added. A and D, enzyme-linked immunosorbent assay (Pierce Colorimetric PKC Assay) to measure total PKC activity.B and F, immunoprecipitation and immunoblot analysis to evaluate phosphorylated serine in MEKK1. The antibodies were specific for MEKK1 and phospho-rylated serine. C and G, results of an IKK immunoprecipitation kinase assay. E, immunoblot analysis of PKC� phosphorylation. An antibody to phosphorylatedPKC� was used.




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NF-�B activation in LPS-stimulated rat peritoneal macro-phages, the cells were treated with PKC inhibitors and theireffects were examined. After pretreatment of macrophageswith 1 �M GF109203X (a PKC inhibitor) for 30 min, LPS-in-duced total PKC activation was inhibited sufficiently (Fig. 3A).Furthermore, phosphorylation of serine in MEKK1 and activa-tion of IKK were decreased (Fig. 3, B and C). However, phos-phorylation of PLC�was unaffected (data not shown). Pretreat-ment of macrophages with Go6976 (10 �M; a PKC� inhibitor)or Go6983 (10 �M; a PKC� inhibitor) for 30 min had no effecton LPS-induced phosphorylation of serine in MEKK1 and onactivation of IKK and NF-�B (data not shown). Nevertheless,after pretreatment with LY379196 (10 �M; a PKC� inhibitor)for 30 min, LPS-induced phosphorylation of serine in MEKK1and activation of IKKwere inhibitedmarkedly (Fig. 3, F andG).Additionally,LPS-inducedtotalPKCactivationandPKC�phos-phorylationwere also inhibited greatly (Fig. 3,D, and E). There-fore, the results suggest that PKC, especially PKC�, participatesin the phosphorylation of serine in MEKK1 in LPS-stimulatedrat peritoneal macrophages.

The PLC Inhibitor Reduced theLPS-induced [Ca2�]i Increase, PKCActivation,MEKK1 Serine Phospho-rylation, and IKK Activation—Theabove results indicate that LPSinduces a transient [Ca2�]i increase,which is due to Ca2� release and asequential Ca2� influx. In non-ex-citable cells, the main source ofintracellular Ca2� is the Ca2� poolin the endoplasmic reticulum; thispool is controlled by the Ins(1,4,5)P3receptor. PLC isoenzymes, includ-ing PLC�, PLC�, and PLC�, catalyzethe hydrolysis of phosphatidylinosi-tol 4,5-biphosphate (PtdIns(4,5)P2)into Ins(1,4,5)P3 and 1,2-diacylglyc-erol (DAG). The present experi-ments revealed that PLC�1 andPLC�2,butnotPLC�3,arephospho-rylated in LPS-stimulated rat peri-toneal macrophages. To furthercharacterize the mechanism ofintracellular Ca2� release, the effectof a PLC inhibitor on LPS-stimu-lated macrophages was examined.After pretreatment with the PLCinhibitor U73122 (1mM) for 30min,LPS-induced phosphorylation ofPLC�1 and PLC�2 was reduced(Fig. 4A). As a result, the transientincrease in [Ca2�]i was inhibitedcompletely (Fig. 4B). Total PKCactivation, phosphorylation of ser-ine in MEKK1, and IKK activationwere also inhibited (Fig. 4, C–E).However, the inhibition of totalPKC activation was not complete

(Fig. 4C). The remainder of PKC activation may be due to cal-cium- and DAG-independent PKCs. Therefore, it can be con-cluded that PLC�1 and PLC�2 regulate intracellular Ca2�

release in LPS-stimulated rat peritoneal macrophages. More-over, it also can be concluded that PLC activation plays animportant role in regulating PKC activation.A PTK Inhibitor Reduced LPS-induced Phosphorylation of

PLC�, [Ca2�]i Increase, PKC Activation, Phosphorylation ofSerine in MEKK1, and IKK Activation—Only after tyrosinephosphorylation can PLC� be activated. To investigate thesignal upstream of PLC, macrophages were pretreated withthe PTK inhibitor genistein (30 �M) for 30 min and then LPSwas added (10 �g/ml). LPS-induced tyrosine phosphoryla-tion of PLC�1 and PLC�2 was inhibited almost completely(Fig. 5A). Meanwhile, the transient increase in [Ca2�]i andPKC activation were inhibited completely (Fig. 5, B and C).Furthermore, phosphorylation of serine in MEKK1 and acti-vation of IKK were also eliminated (Fig. 5, D and E). There-fore, the results indicate that tyrosine phosphorylation ofPLC�1 and PLC�2 is PTK-dependent. They also suggest that

FIGURE 4. A PLC inhibitor (U73122) decreased LPS-induced phosphorylation of PLC�, the [Ca2�]iincrease, PKC activation, phosphorylation of serine in MEKK1, and IKK activation in rat peritoneal mac-rophages. Macrophages were not pretreated (�) or were pretreated (�) with U73122 (1 mM) for 30 min, andthen LPS (10 �g/ml) was added. A, immunoblot analysis of phosphorylation of PLC�1 and PLC�2. An antibodyto phosphorylated PLC�1 and PLC�2 was used. B, measurement of [Ca2�]i in an individual LPS-stimulatedmacrophage after pretreatment with U73122. C, enzyme-linked immunosorbent assay (Pierce ColorimetricPKC Assay) to measure the total PKC activity. D, immunoprecipitation and immunoblot analysis to detectphosphorylated serine in MEKK1. The antibodies were specific for MEKK1 and phosphorylated serine. E, resultsof an IKK immunoprecipitation kinase assay.




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PTK is involved in regulating other detected effectors inLPS-stimulated rat peritoneal macrophages.A MEKK Inhibitor Reduced IKK Activation, NF-�B Activa-

tion, iNOS Expression, and TNF-� Production—The results ofthe present study demonstrated that the phosphorylation ofserine in MEKK1 is PKC�-dependent in LPS-stimulated ratperitoneal macrophages (Fig. 3). However, the question abouthowMEKK1 transduces the signal downstream is unanswered.To investigate the role of MEKK in the signaling pathway ofNF-�B activation, macrophages were pretreated with theMEKK inhibitor PD98059 (20 �M; a MEKK1-specific inhibitoris not available) for 30 min. After pretreatment, LPS-inducedIKK activationwas decreased (Fig. 6A). In addition,NF-�B acti-vation, iNOS expression, and TNF-� production were alsoinhibited (Fig. 6, B–D). These results indicate that MEKKs par-ticipate in the regulation of IKK activation in LPS-stimulatedrat peritoneal macrophages.

DISCUSSION

The signaling pathway for LPS-induced production of pro-inflammatory mediators in macrophages has been studied for

many years. TLR4 associates withseveral kinases and adaptors totransduce signals downstream viaTRAF6. Sequentially, MAPKs areactivated by a three-tiered cascadeof kinases, and NF-�B is activatedafter IKK phosphorylates I�B�.However, the question about whatkinds of signaling molecules con-nect TRAF6 to IKK andMAPKs hasremained unanswered (6, 7, 27–29).In the present study,we investigatedthe characteristics, mechanisms,and roles of the transient increase in[Ca2�]i and PKC activation in thesignaling pathway for LPS-inducedNF-�B activation, iNOS expression,and TNF-� production in rat peri-toneal macrophages. We system-atically monitored LPS-induceddynamic changes, such as those intyrosine phosphorylation of PLC, thetransient increase in [Ca2�]i, activa-tion of PKC, phosphorylation of ser-ine in MEKK1, activation of IKK,accumulation of phosphorylatedI�B� in the cytoplasm, transloca-tion of p50/p65 in the nucleolus,activation of NF-�B, expression ofiNOS, and production of TNF-�(Fig. 1).As a second messenger, intracel-

lular Ca2� is essential for many cel-lular responses. In the presentstudy, LPS aroused a rapid transientincrease in [Ca2�]i in rat peritonealmacrophages. When extracellular

Ca2� was removed, the initial rate of [Ca2�]i increase did notdiffer from that seen when extracellular Ca2� was present, butthe peak in [Ca2�]i was lower when extracellular Ca2� wasabsent (Fig. 1). Thus, it indicates that the LPS-induced transientincrease in [Ca2�]i begins with the release of Ca2� from intra-cellular Ca2� pools in rat peritoneal macrophages, and thisrelease may be followed by Ca2� influx from the extracellularspace. In non-excitable cells such as rat peritoneal macro-phages, Ca2� is released mainly through the opening of theIns(1,4,5)P3 receptor in the endoplasmic reticulumwhen intra-cellular Ins(1,4,5)P3 increases (30). Ins(1,4,5)P3 is produced inthe cytoplasm when active PLC catalyzes the hydrolysis ofPtdIns(4,5)P2 into Ins(1,4,5)P3 andDAG (19). ThePLC family iscomposed of several PLC isoenzymes. One subfamily includesPLC� and PLC, both of which are activated by the receptor-coupledGq protein in themembrane (31, 32). Another subfam-ily is PLC�, whose tyrosine phosphorylation depends on theactivation of PTK (33, 34), but it is unknown which type(s) ofPLC isoenzymes that mediate the transient increase in [Ca2�]iare activated in LPS-stimulated rat peritoneal macrophages.Results from the present study provide insight into this issue.

FIGURE 5. A PTK inhibitor (genistein) reduced LPS-induced phosphorylation of PLC�1 and PLC�2, the[Ca2�]i increase, PKC activation, phosphorylation of serine in MEKK1, and IKK activation in rat perito-neal macrophages. Macrophages were not pretreated (�) or were pretreated (�) with genistein (30 �M) for 30min, and then LPS (10 �g/ml) was added. A, immunoblot analysis of phosphorylated PLC�1 and PLC�2. B,measurement of [Ca2�]i in an individual LPS-stimulated rat peritoneal macrophage after pretreatment withgenistein. C, enzyme-linked immunosorbent assay (the Pierce Colorimetric PKC Assay) to measure total PKCactivity. D, immunoprecipitation and immunoblot analysis of phosphorylated serine in MEKK1. The antibodieswere specific for MEKK1 and phosphorylated serine. E, results of an IKK immunoprecipitation kinase assay.




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First, in the present study, the phosphorylation of a member ofthe PLC� subfamily, PLC�3, was not detected, whereas tyro-sine residues in PLC�1 and PLC�2 were phosphorylated sub-stantially (Fig. 1). Second, when rat peritoneal macrophageswere pretreated with the PTK inhibitor genistein, LPS-inducedphosphorylation of PLC�1 and PLC�2 was inhibited com-pletely, and the LPS-induced transient increase in [Ca2�]i wasalso absent (Fig. 5). Third, the PLC inhibitor U73122 bluntedthe transient increase in [Ca2�]i (Fig. 4). Fourth, the LPS-acti-vated receptor TLR4 is not coupled to the Gq protein in mac-rophages. Moreover, no report has revealed that a Gq protein-coupled receptor is activated in LPS-stimulated macrophages.Fifth, there is no evidence demonstrating that PLC�, -�, -�, and- are activated in LPS-stimulated macrophages. Takentogether, the present information suggests that PTK-mediatedphosphorylation of PLC�1 and PLC�2 regulates Ca2� release.The present results indicate that PTKmediates activation of

PLC�1 and PLC�2.When a PTK inhibitor (genistein) was used,there was complete inhibition of a series of biological eventsinduced by LPS, including phosphorylation of PLC�1 andPLC�2, a transient increase in [Ca2�]i, total PKC activation,phosphorylation of serine in MEKK1, and activation of IKK

(Fig. 5). PTK also affects a series of biological effectors in ratperitoneal macrophages. However, the specific PTK that medi-ates phosphorylation of PLC�1 and PLC�2 in rat peritonealmacrophages has not been identified. Recently, the activationof some non-receptor PTKs was detected in the TLR4 signalingpathway. The tyrosine kinase Btk is involved in TLR4-mediatedactivation of NF-�B. In LPS-stimulated macrophages, Btk reg-ulates NF-�B activation and participates in the production ofseveral pro-inflammatory mediators (35–37).According to the present results, LPS-induced transient

increase in [Ca2�]i is due to intracellular Ca2� release andextracellular Ca2� influx. When extracellular or intracellularCa2� was chelated, phosphorylation of PKC� and PKC� waseliminated almost completely, whereas phosphorylation ofPKC� and PKC was not affected obviously. Furthermore, totalPKC activity was inhibited greatly (Fig. 2). Because activatedPLC� can catalyze PtdIns(4,5)P2 hydrolysis to produce DAGand Ins(1,4,5)P3, we believe that these results suggest that phos-phorylation of PLC� and the transient increase in [Ca2�]i leadto Ca2�- and DAG-dependent activation of PKC� and PKC�.The activation of other PKCs (PKC� and PKC) is DAG-de-pendent in LPS-stimulated rat peritoneal macrophages.When phosphorylation of PLC�1 and PLC�2 was inhibited

completely by U73122, total PKC activation was not eliminatedcompletely (Fig. 4). Nevertheless, when the PTK inhibitorgenistein inhibited phosphorylation of PLC�1 and PLC�2 com-pletely, it also eliminated total PKC activation (Fig. 5). Theseresults suggest that total PKC activationmay depend on factorsother than PLC. PTKmay contribute to some Ca2�- and DAG-independent activation of PKC in rat peritoneal macrophages.Recent reports have revealed that LPS can activate atypicalPKCs in macrophages (38).In other types of cells, MEKK1 is regulated by particular

kinases such as the germinal center kinase andNF-�B-inducingkinase (39, 40). Until now, no report has suggested that PKC�regulates phosphorylation of serine inMEKK1 in rat peritonealmacrophages. In our study, the PKC inhibitor GF109203Xdecreased LPS-induced phosphorylation of serine in MEKK1and activation of IKK and NF-�B. Moreover, the PKC� inhibi-tor (LY379196) reduced this serine phosphorylation dramati-cally (Fig. 3), but other PKC isoenzyme inhibitors (Go6976,which inhibits PKC�, and Go6983, which inhibits PKC) hadno obvious effect (data not shown). Therefore, these resultssuggest that PKC� participates in phosphorylation of serine inMEKK1 in LPS-stimulated rat peritoneal macrophages.TheMEKK family includes kinases such asMEKK1,MEKK2,

and MEKK3 (41–43). In some cells, MEKK1 and MEKK3 areinvolved in the activation of IKK in the signaling pathway forIL-1 receptor/TLR4-mediated activation of NF-�B (44, 45).However, similar studies of peritoneal macrophages have beenfew. In our study, we were unable to detect a relationshipbetween MEKK1 alone and IKK because no MEKK1-specificinhibitor was available. When the MEKK inhibitor PD98059was used, LPS-induced activation of IKK and NF-�B wereinhibited partially (Fig. 4). A possible reason for this result maybe that kinases other than MEKKs regulate IKK activation. Forexample, TAK1, phosphoinositide 3-kinase, andNF-�B-induc-ing kinase regulate IKK activation (26, 46–50). Thus, these

FIGURE 6. A MEKK inhibitor (PD98059) decreased LPS-induced IKK andNF-�B activation, iNOS expression, and TNF-� production in rat perito-neal macrophages. Macrophages were not pretreated (�) or were pre-treated (�) with PD98059 (20 �M) for 30 min, and then LPS (10 �g/ml) wasadded. A, results of an IKK immunoprecipitation kinase assay. B, results of adual luciferase assay to evaluate the extent of NF-�B activation. C, results ofimmunoblot analysis to evaluate iNOS expression. The antibody was specificfor iNOS. D, results of TNF-� assays. The filled circle indicates the values for thecontrol cells; the filled triangle, the values for cells treated with LPS alone; andthe filled square, the values for cells treated with LPS plus PD98059.




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results suggest that MEKK participates in, but is not the soleone responsible for IKK activation in LPS-stimulated rat peri-toneal macrophages.An important transcription factor for the expression of

iNOS, ILs, and TNF-�, NF-�B is a p50/p65 dimer in macro-phages. After binding with I�B�, NF-�B is inhibited. WhenI�B� is phosphorylated by IKK, the p50/p65 dimer dissociatesfrom the complex and relocates to the nucleolus; this processleads toNF-�B activation (14, 15). In the present study, after theactivation of IKK, therewas an increase in phosphorylated I�B�in the cytoplasm, p65 in the nucleolus, and NF-�B activation(Fig. 1).In general, LPS evokes a series of events in rat peritoneal

macrophages. The above analysis of these cells after treatment

with kinase or enzyme inhibitors revealed a series of biologicalevents upon which we propose the components and theirsequence in the Ca2�- and PKC-dependent signaling pathwayfor NF-�B activation (Fig. 7). First, LPS acts with TLR4 andmediates PTK activation in macrophages. Second, PTK mayinduce PLC�1 and PLC�2 phosphorylation, which leads toPtdIns(4,5)P2 hydrolysis and thus production of DAG andIns(1,4,5)P3.With the increase of Ins(1,4,5)P3 in the cytoplasm,intracellular pools release Ca2�, and this release may be fol-lowed byCa2� influx fromextracellular spaces. Activated PLC�and the transient increase in [Ca2�]i activate PKC isoenzymes.Next, PKC� regulates the phosphorylation of serine inMEKK1.Activated MEKK participates in IKK activation, and activatedIKK in turn activates NF-�B by phosphorylating I�B� andinducing the translocation of the p65/p50 dimer into the nucle-olus. Finally, the expression of iNOS and the production ofTNF-� are elevated.The PLC inhibitor, the PKC inhibitor, and the MEKK inhib-

itor did not inhibit LPS-induced IKK activation completely, butthe PTK inhibitor eliminated all of these events. Therefore, it isreasonable to regard this LPS-activated pathway as oneinvolved in NF-�B activation. In fact, LPS activates IKK andNF-�B by a complex signaling network. Meanwhile, LPS alsoactivates MAPKs. Then, activator protein-1, ATF, and othertranscription factors are activated. Therefore, we hypothesizethat the cooperation of these signaling pathways may beresponsible for the activation of transcription factors that pro-mote the expression and release of inflammatory mediators.

Acknowledgments—We thank Drs. Changyan Li, Fuchu He, andXiaoming Yang for providing help in conducting the experiments. Wealso thank Drs. Zongjie Cui and Julia Cay Jones for reading throughand helping with rewriting the manuscript.

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Xueyuan Zhou, Wenxiu Yang and Junying LiMacrophages

Production in Lipopolysaccharide-stimulated Rat PeritonealαFactor-Activation, Inducible Nitric-oxide Synthase Expression, and Tumor Necrosis

Bκ- and Protein Kinase C-dependent Signaling Pathway for Nuclear Factor-2+Ca

doi: 10.1074/jbc.M602739200 originally published online August 21, 20062006, 281:31337-31347.J. Biol. Chem.

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ca - and protein kinase c-dependent signaling pathway for

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